Recent studies in our laboratory and elsewhere have demonstrated that, in mammalian cells with acquired resistance to antifolates associated with overproduction of target enzyme dihydrofolate reductase (DHFR) via a gene amplification process, there may be distinctive chromosomal abnormalities specifically related to the resistant phenotype. We propose to probe, by techniques of molecular hybridization of DNA to metaphase chromosomes in situ, the location and chromosomal organization of amplified DHFR-encoding genes. As we reported initially, Chinese hamster fibroblast-like cells with high levels of resistance to antifolate contain a long, homogeneously staining region (designated "HSR") on a metaphase chromosome as revealed by the trypsin-Giemsa banding technique. Direct confirmation that HSRs contain amplified DHFR genes was obtained by Nunberg et al., 1978, and direct confirmation that our highly resistant lines contain amplified DHFR genes has been obtained by Dr. P. W. Melera. We have extensively analyzed our large series of sublines selected with methotrexate or methasquin. In sublines with only a 3- to 50-fold increase in target enzyme activity there are long chromosome regions that are banded but that are distinctly abnormal in band pattern. The abnormally banding regions as well as HSRs are preferentially localized to the long (q) arm of Chinese hamster chromosome 2, suggesting that the abnormally banding regions in low resistance lines are in some ways precursors to the HSRs of highly resistant lines. Whether the abnormal regions result from and thus signify DHFR gene amplification is an important question. Cytological experiments will be carried out with kinetically purified cDNA complementary to partially purified DHFR mRNA and cloned full-length cDNA copy of the specific mRNA; probe will be supplied by Dr. P. W. Melera. Specifically, we propose to: 1) verify the HSR location of DHFR genes in highly resistant sublines with the use of species-specific cDNA probe for hybridization to metaphase chromosomes in situ; 2) determine whether there are DHFR gene clusters on chromosome 2q in the abnormally banded cells with lower levels of resistance; 3) determine detectability levels of DHFR genes by comparisons of the cytological data with gene titration data obtained by Dr. Melera; and 4) utilize the Chinese ham (Text Truncated - Exceeds Capacity)